CN106404864B - Plant methyl jasmonate detection method based on microelectrode biosensing technology - Google Patents
Plant methyl jasmonate detection method based on microelectrode biosensing technology Download PDFInfo
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- CN106404864B CN106404864B CN201610964019.7A CN201610964019A CN106404864B CN 106404864 B CN106404864 B CN 106404864B CN 201610964019 A CN201610964019 A CN 201610964019A CN 106404864 B CN106404864 B CN 106404864B
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- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
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Abstract
The present invention provides a kind of plant methyl jasmonate detection method based on microelectrode biosensing technology, is microelectrode biosensor through the invention, realizes the on-line checking of methyl jasmonate in plant.It is small, easy to operate, easy to carry that this method has the characteristics that accuracy height, sample destroy, and on time and space aspects, realizes that the synchronous of methyl jasmonate information obtains in plant, provides technical support for plant research.Compared with traditional bioassay, chemical detection method, this method sample pre-treatments are simple, and detection position is injured without in vitro, micro- damage, without essence, substantive damage will not be caused to plant, testing result is reliable, high sensitivity.
Description
Technical field
The present invention relates to microelectrode biosensing technologies, specifically, being related to a kind of based on microelectrode biosensing technology
Plant methyl jasmonate detection method.
Background technique
Methyl jasmonate is widely present in a kind of growth regulatory substance of plant, has a variety of physiological effects, as can mentioning
High seedling can stimulate the synthesis of antifungal protein to starting or acceleration and defense response the resistivity of high/low temperature arid
Related secondary metabolism inhibits Growth and Differentiation, hestening rooting ripening and senscence etc..Currently, the physiology important due to methyl jasmonate
Effect and relatively low content in plant, the detection of methyl jasmonate are more and more taken seriously.
Method currently used for the methyl jasmonate in detection plant is mainly chromatography, including gas chromatography mass spectrometry, liquid matter
Combination, high performance liquid chromatography, capillary liquid chromatography etc. also have enzyme linked immunosorbent detection technology.Instrument used in these methods
Majority operation is complicated, and higher cost, the period is longer, and sample is destructive big, cannot achieve plant growth on-line checking.Electrochemical credit
Analysis method is suitble to the qualitative, quantitative inspection that plant living body is online because of the advantages that its is easy to operate, response is rapid and sensitivity is higher the most
It surveys.
Summary of the invention
The object of the present invention is to provide a kind of plant methyl jasmonate detection side based on microelectrode biosensing technology
Method.
In order to achieve the object of the present invention, present invention firstly provides a kind of microelectrode biosensor, the microelectrode is in three
Pyramid, long 1-10cm, the long 2-20mm of tip portion, the side length of other end cross-sectional triangle are that 1-15mm is (preferably another
End cross section is equilateral triangle)；The basal electrode material of the microelectrode is by the silicon wafer of insulation processing, stainless steel or to have
Machine glass etc.；On each face of the substrate, one end is provided with the element for connecting electrochemical workstation, and the other end is inspection
The electrode material of survey is connected between the element and electrode material by conducting wire；The microelectrode has three-electrode system, packet
Reference electrode containing Ag/AgCl, platinum is to electrode and the metal working electrode modified by platinum, graphene and perfluorinated sulfonic resin.
The microelectrode biosensor can be prepared as follows to obtain:
1) platinum is prepared in substrate using micro-electromechanical technology (MEMS) to make electrode, Ag/AgCl reference electrode and metal working
Electrode；
2) metal working electrode is polished on abrasive paper for metallograph with alumina powder, is carried out respectively with deionized water, ethyl alcohol
Microelectrode, is then placed in 0.5M dilution heat of sulfuric acid, cyclic voltammetry scan is carried out under 0-1.5V current potential, is obtained by ultrasonic cleaning
Typical cyclic voltammetric spectrogram, it is ensured that electrode surface is clean；
3) 1mg/ml graphene oxide and 1mM chloroplatinic acid mixed solution are prepared, the microelectrode is immersed in above-mentioned solution,
Electrochemical reduction deposition 0.5~2h (preferably 1h) is carried out by timing Amperometric under -0.8V voltage, is then washed with deionized water
Only, the graphene/platinum electrode modified；
4) by the perfluor sulfoacid resin solution of concentration 0.005~0.1% (preferably 0.02%) drop coating in the graphene/platinum
On electrode, drying and forming-film (drying temperature is generally below 100 DEG C, meets the condition of drying and forming-film), is then used at 50 DEG C
Deionized water is cleaned, and the metal working electrode modified by platinum, graphene and perfluorinated sulfonic resin is obtained, to complete the microelectrode
The preparation of biosensor.
The present invention also provides application of the microelectrode biosensor in plant jasmonate acid methyl esters on-line checking.It is
The microelectrode biosensor is inserted into plant tissue to be measured, connection electrochemical workstation carries out cyclic voltammetry scan, real
Online methyl jasmonate detection now is carried out to plant tissue.
The methyl jasmonate standard solution that various concentration is chosen before detection carries out differential pulse voltammetry scanning, according to jasmine
Sour methyl acetate concentrations and current relationship obtain microelectrode and detect methyl jasmonate working curve；Testing conditions are as follows: current potential 0.9-1.4V,
Current potential increment 0.004V, amplitude 0.05V, pulse width 0.02s, pulse period 0.5s, quiescent time 20s.
Wherein, the methyl jasmonate standard solution of the various concentration is prepared using the 10mM phosphate buffer of pH6.0.
The microelectrode biosensor lowest detection is limited to 0.5 μm of ol/L, and the range of linearity is 1-100 μM, jasmonic first
The linear equation of ester solution concentration and response peak current are as follows: I=0.682+1.71C, relative coefficient 0.9847；Wherein, I table
Show electric current, unit is μ A, and C indicates that concentration, unit are μM.
Test object of the invention includes the positions such as root, stem, leaf or the fruit of plant, and insertion depth is 0.5cm or so.Inspection
Survey the different times that target material is plant growth, such as the stem of wheat seedling.
Application above-mentioned is corrected the microelectrode biosensor before further including the steps that detection, specific method
It is as follows: microelectrode is cleaned, then respectively with the methyl jasmonate solution of two parts of normal concentrations (such as 5 μM and 50 μM) into
Row electrochemistry calibration, if working curve and slope of standard curve deviation within 15%, determine that electrode can work normally.
Differential pulse voltammetry scanning, testing conditions are as follows: current potential 0.9-1.4V, current potential increment are carried out to sample after correction
0.004V, amplitude 0.05V, pulse width 0.02s, pulse period 0.5s, quiescent time 20s.
Electrochemical workstation is connected, after system activation 20min, stable operation 1min-10min acquires current data and substitutes into line
Property equation, obtains the concentration of methyl jasmonate in plant tissue.
The online methyl jasmonate detection method of a kind of plant based on microelectrode biosensing technology provided by the invention, should
It is small, easy to operate, easy to carry that method has the characteristics that accuracy height, sample destroy, and on time and space aspects, realizes and plants
The synchronous of methyl jasmonate information obtains in object, provides technical support for plant research.With traditional bioassay, change
It learns detection method to compare, this method sample pre-treatments are simple, and detection position is injured without in vitro, micro- damage, without essence, will not be to plant
Object causes substantive damage, and testing result is reliable, high sensitivity.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of the microelectrode biosensor prepared in the embodiment of the present invention 1；Wherein, the face A-work
Electrode, the face B-reference electrode, the face C-is to electrode.
Fig. 2 is that the working electrode of microelectrode biosensor in the embodiment of the present invention 1 modifies schematic diagram.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
Used in the conventional means that are well known to those skilled in the art of technological means, raw materials used is commercial goods.
1 microelectrode biosensor of embodiment and preparation method thereof
The present embodiment prepares microelectrode, micro- electricity using micro-electromechanical technology in the stainless steel base Jing Guo insulation processing
Pole is in triangular pyramid, and total length 5cm, tip portion length is 5mm, and other end cross section is equilateral triangle, and side length is
3mm；On each face of the substrate, one end is provided with the element for connecting electrochemical workstation, and the other end is that detection is used
Electrode material, be connected between the element and electrode material by conducting wire；The microelectrode has three-electrode system, includes
Ag/AgCl reference electrode, platinum is to electrode and metal working electrode (Fig. 1 and the figure modified by platinum, graphene and perfluorinated sulfonic resin
2)。
The metal working electrode by the modification of platinum, graphene and perfluorinated sulfonic resin can be prepared as follows to obtain:
1, metal working electrode is polished on abrasive paper for metallograph with alumina powder, is surpassed respectively with deionized water, ethyl alcohol
Microelectrode, is then placed in 0.5M dilution heat of sulfuric acid, cyclic voltammetry scan is carried out under 0-1.5V current potential, obtains allusion quotation by sound cleaning
The cyclic voltammetric spectrogram of type, it is ensured that electrode surface is clean；
2,1mg/ml graphene oxide and 1mM chloroplatinic acid mixed solution are prepared, the microelectrode is immersed in above-mentioned solution,
Electrochemical reduction is carried out by timing Amperometric under -0.8V voltage and deposits 1h, is then washed with deionized water, is modified
Graphene/platinum electrode；
3, it by the perfluor sulfoacid resin solution drop coating of concentration 0.02% on the graphene/platinum electrode, is done at 50 DEG C
Dry film forming, is then washed with deionized water, and obtains the metal working electrode modified by platinum, graphene and perfluorinated sulfonic resin, thus
Complete the preparation of the microelectrode biosensor.
Embodiment 2 carries out real-time online detection to plant jasmonate acid methyl esters using microelectrode biosensor
1, experimental subjects
It is wheat breed ' the agricultural university 6812 ' of Beijing City Agriculture and Forestry Institute plantation to measuring plants, chooses two plants of wheat seedlings
Stem is as detection sample.
After cleaning, microelectrode prepared by embodiment 1 is inserted into sample to be tested stem, starts Electrochemical Detection.
2, laboratory apparatus and consumptive material
Laboratory apparatus uses Shanghai Chen Hua electrochemical workstation 760e.
3, experimental method
(1) drafting of standard curve:
The methyl jasmonate standard solution that various concentration is chosen before detection carries out differential pulse voltammetry scanning, according to jasmine
Sour methyl acetate concentrations and current relationship obtain microelectrode and detect methyl jasmonate working curve；Testing conditions are as follows: current potential 0.9-1.4V,
Current potential increment 0.004V, amplitude 0.05V, pulse width 0.02s, pulse period 0.5s, quiescent time 20s.
Wherein, the methyl jasmonate standard solution of the various concentration is prepared using the 10mM phosphate buffer of pH6.0.
The microelectrode biosensor lowest detection is limited to 0.5 μm of ol/L, and the range of linearity is 1-100 μM, jasmonic first
The linear equation of ester solution concentration and response peak current are as follows: I=0.682+1.71C, relative coefficient 0.9847；Wherein, I table
Show electric current, unit is μ A, and C indicates that concentration, unit are μM.
(2) experimental procedure:
The microelectrode biosensor is corrected before detection, the method is as follows: microelectrode is cleaned, is then divided
Electrochemistry calibration is not carried out with the methyl jasmonate solution of two parts of normal concentrations (5 μM and 50 μM), if working curve and standard
Slope of curve deviation determines that electrode can work normally within 15%.
Differential pulse voltammetry scanning, testing conditions are as follows: current potential 0.9-1.4V, current potential increment are carried out to sample after correction
0.004V, amplitude 0.05V, pulse width 0.02s, pulse period 0.5s, quiescent time 20s.
Electrochemical workstation is connected, after system activation 20min, stable operation 1min-10min acquires current data and substitutes into line
Property equation, obtains the concentration of methyl jasmonate in plant tissue.
4, experimental evaluation result
Using HPLC method and the methyl jasmonate content ratio of on-line checking analysis Liang Zhu wheat seedling stem of the invention
It relatively the results are shown in Table 1.As can be seen that this method testing result is reliable, methyl jasmonate may be implemented by electrochemical workstation and exist
The analysis of line dynamic detection provides new method for living body online study plant jasmonate acid methyl ester content, realizes to existing detection hand
The supplement of section.
The methyl jasmonate test result (unit, μ g/g) of 1 wheat seedling stem of table
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. being used for the microelectrode biosensor of plant jasmonate acid methyl esters on-line checking, which is characterized in that the microelectrode is in three
Pyramid, long 1-10cm, the long 2-20 mm of tip portion, the side length of other end cross-sectional triangle are 1-15mm；Micro- electricity
The basal electrode material of pole is the silicon wafer, stainless steel or organic glass by insulation processing；On each face of the substrate, one
End is provided with the element for connecting electrochemical workstation, and the other end is the electrode material of detection, the element and electrode material
It is connected between material by conducting wire；The microelectrode has three-electrode system, includes Ag/AgCl reference electrode, platinum to electrode and
The metal working electrode modified by platinum, graphene and perfluorinated sulfonic resin；
The preparation method of the microelectrode biosensor the following steps are included:
1) platinum is prepared to electrode, Ag/AgCl reference electrode and metal working electrode in substrate using micro-electromechanical technology；
2) metal working electrode is polished, is cleaned by ultrasonic respectively with deionized water, ethyl alcohol, microelectrode is then placed in 0.5
In M dilution heat of sulfuric acid, cyclic voltammetry scan is carried out under 0-1.5 V current potential, obtains typical cyclic voltammetric spectrogram, it is ensured that electrode
Surface is clean；
3) 1 mg/ml graphene oxide and 1 mM chloroplatinic acid mixed solution are prepared, the microelectrode is immersed in above-mentioned solution,
Electrochemical reduction is carried out by timing Amperometric under -0.8V voltage and deposits 0.5 ~ 2h, is then eluted with water, the stone modified
Black alkene/platinum electrode；
4) by the perfluor sulfoacid resin solution drop coating of concentration 0.005 ~ 0.1% on the graphene/platinum electrode, drying and forming-film, so
After be eluted with water, the metal working electrode modified by platinum, graphene and perfluorinated sulfonic resin is obtained, so that it is raw to complete the microelectrode
The preparation of object sensor.
2. application of the microelectrode biosensor described in claim 1 in plant jasmonate acid methyl esters on-line checking.
3. application according to claim 2, which is characterized in that insert microelectrode biosensor as claimed in claim 1 or 2
Enter in plant tissue to be measured, connection electrochemical workstation carries out cyclic voltammetry scan, realizes and carries out online jasmine to plant tissue
Sour methyl esters detection.
4. application according to claim 3, which is characterized in that the methyl jasmonate standard for choosing various concentration before detection is molten
Liquid carries out differential pulse voltammetry scanning, obtains microelectrode according to methyl jasmonate concentration and current relationship and detects methyl jasmonate
Working curve；Testing conditions are as follows: current potential 0.9-1.4 V, 0.004 V of current potential increment, 0.05 V of amplitude, 0.02 s of pulse width,
0.5 s of pulse period, 20 s of quiescent time.
5. application according to claim 4, which is characterized in that the microelectrode biosensor lowest detection is limited to
0.5umol/L, range of linearity 1-100uM, the linear equation of methyl jasmonate solution concentration and response peak current are as follows: I=
0.682+1.71C, relative coefficient 0.9847；Wherein, I indicates electric current, and unit is μ A, and C indicates that concentration, unit are μM.
6. according to the described in any item applications of claim 2-5, which is characterized in that test object includes the root, stem, Ye Huo of plant
Fruit.
7. application according to claim 6, which is characterized in that test object is the stem of wheat seedling.
8. according to the described in any item applications of claim 2-5, which is characterized in that further include that detection is preceding to the microelectrode biology
The step of sensor is corrected, the specific method is as follows: cleaning to microelectrode, then respectively with the jasmine of two parts of normal concentrations
Jasmine acid methyl ester solution carries out electrochemistry calibration, if working curve and slope of standard curve deviation within 15%, determine that electrode can
It works normally.
9. application according to claim 8, which is characterized in that carry out differential pulse voltammetry scanning, inspection to sample after correction
Survey condition are as follows: current potential 0.9-1.4 V, 0.004 V of current potential increment, 0.05 V of amplitude, 0.02 s of pulse width, pulse period 0.5
S, 20 s of quiescent time.
10. application according to claim 9, which is characterized in that connection electrochemical workstation, after system activation 20min, surely
Surely operation 1min-10min acquisition current data substitutes into linear equation, obtains the concentration of methyl jasmonate in plant tissue.
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| CN108398476B (en) * | 2017-12-29 | 2020-04-10 | 北京农业智能装备技术研究中心 | Biosensor for in-vivo detection of salicylic acid and construction method and application thereof |
| CN108303453B (en) * | 2017-12-29 | 2020-04-10 | 北京农业智能装备技术研究中心 | Sensor for simultaneously detecting salicylic acid and indoleacetic acid in living body and construction method thereof |
| CN113786853B (en) * | 2021-08-06 | 2023-01-17 | 中国科学院化学研究所 | Monoatomic catalyst, preparation method thereof, microelectrode, preparation method thereof and application thereof |
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